Centrifugation is a technique used to process whole blood in order to separate the blood into its various components. To reduce personal contact with blood products and reduce cross-contamination between different blood sources, the centrifugal apparatus can be fitted with a disposable plastic vessel through which the blood is circulated. The vessel is fitted into a centrifuge fixture that is driven by a motor. An exemplary vessel is a circumferential separation channel having several outlets positioned at different radial positions within the channel in order to remove blood components which have been separated by the centrifuge into stratified layers of differing density. Red blood cells (RBC) being the most dense of the components are stratified within the channel at the most radially outward location whereas the stratified layer of plasma is the least dense component and therefore the most radially inward layer. A relatively thin layer called the buffy coat contains white blood cells and platelets and is located between the red blood cell layer and the plasma layer. Within the buffy coat the platelets are stratified toward the plasma while the white blood cells are stratified toward the red blood cells. Depending on centrifuge speed, platelets may also be dispersed within the plasma.
The disposable plastic vessel which is fitted into a rotating fixture within the centrifuge is connected to the blood source and to collection reservoirs through a disposable tubing set. In that manner, the centrifuge equipment itself is kept out of contact with blood and the disposable tubing set and separation channel are discarded after one procedure. The source of blood can be whole blood flowing directly from a donor or patient, or it can be previously donated bone marrow or blood.
Blood components may be collected from a patient, stored and perhaps frozen, and reinfused into the patient days or even years later. The mononuclear cell portion of white blood cells is sometimes collected, stored in the above manner, and reinfused into the patient for the treatment of diseases such as cancer. There are obvious advantages to returning blood components from the patient's own blood rather than using the blood of a donor. It is generally agreed that the safest blood a person can receive is his or her own blood (autologous blood). The use of autologous blood reduces the risk of exposure to transfusion transmitted disease and febrile/allergic transfusion reactions. To accomplish the collection of white blood cells (WBC), an apheresis system has been developed for harvesting them from the buffy coat. In particular, the mononuclear cell (MNC) portion of WBCs are harvested including lymphocytes, monocytes, progenitor cells, and stem cells. In this document the designations WBCs and MNCs are usually used interchangeably. Efficient equipment for collecting MNCs is described in U.S. Pat. No. 4,647,279. However, even with efficient equipment, the collection of mononuclear cells is difficult since they make up only a small fraction of the total blood volume. For a patient of normal size with a normal MNC count, the total volume of MNCs may be about 1.5 milliliters, that is about 0.03% of the total blood volume. As a consequence, when whole blood is centrifuged, only a very thin MNC layer appears between the red blood cell and plasma layers.
The thin MNC layer presents a challenge when attempting an MNC harvest. Because the MNC fraction of whole blood is so small, the equipment referred to above includes a barrier positioned in the channel upstream of the RBC exit port. MNCs are accumulated at the barrier with a WBC collection port placed in front of the barrier. The fraction collected through the WBC collection port is actually a mixture of WBCs, platelets, plasma and RBCs. In collection procedures, the color of the collected fraction may be monitored in order to adjust the blood inflow and plasma outflow rates, if necessary, manually or automatically, to fine tune the position of the MNC layer so that the MNC layer corresponds in position with the WBC collection port. Usually, an operator makes very fine adjustments to the speed of the plasma pump in order to position the MNC layer properly for collection. If manual monitoring is used, the operator judges the position of the MNC layer according to the color of the fluid leaving the collection channel, and adjustments are made to provide the desired color in the collect port. If, for example, the operator begins to observe a reddish tint, the presence of RBCs are signified in the collect line. In such case, there is a need to increase the amount of plasma in the separation channel so that the RBC layer can be lowered. That can be accomplished by reducing plasma pump speed. Fine control is provided over the speed of the plasma pump such that adjustments may be made in collect volume on the order of one tenth milliliter per minute. Even though small changes are possible in the speed of the pump, it is not unusual for a change in plasma pump speed to over or under-correct, necessitating further change in pump speed. As a consequence, the interface positioning system, manual or automatic, can be involved in a vibratory chasing of the correct interface position with the result of decreased efficiency and purity in collecting the MNC layer. A further problem is that after each change in pump speed the process requires a period of time for the change to take effect, that is, for the new interface position to become established. Attempts have been made to use optical monitoring equipment to judge the opacity of the collect volume and automatically adjust plasma pump speed. However, such techniques designed to automate the system are also subject to oscillations around the control point and generally provide little improvement over the system when it is operated manually. Basically, all of these problems result from the fact that the target species is sparse and forms a very thin stratified layer which is difficult to harvest separately from other components, and because of the relatively low response of the interface to changes in the flow rate.
Because of the difficulty in properly positioning and maintaining the interface, a relatively wide band of volume is collected from the WBC port so that there is an assurance that the thin white blood cell layer has been collected. By collecting a wider band, however, a considerable amount of plasma, platelets, or red blood cells are also collected together with the white blood cells. Such a technique is efficient in the sense that it collects most of the stratified white cells, but it is low in purity. Also, the volume of collection is increased over what is needed. The goals of high MNC yield or efficiency and a low collection volume of high purity are somewhat mutually exclusive since it is difficult to extract only the thin stratified layer of white blood cells. Generally, volume and purity are sacrificed in favor of collection efficiency.
To further explain and illustrate, WBCs are comprised of mononuclear cells and polymorphonuclear cells (PMNs including granulocytes). Granulocytes are normally a small sub-population of WBCs in healthy people but grow to a more significant sub-population when the body reacts to disease. When whole blood is centrifuged, depending on centrifuge speed, the thin buffy coat layer is itself stratified into a still thinner layer of MNCs and a thin layer of platelets. The granulocytes are found in the buffy coat tending more toward the RBC layer and are also found in significant populations within the RBC layer. When the needs of a patient make it advisable to harvest granulocytes, a drug is generally provided to the patient which enables the granulocytes to migrate from the RBC layer into the buffy coat as a thin layer between the RBCs and the MNCs. In harvesting granulocytes, it has been necessary to also collect MNCs since the layers are too thin to be harvested separately. A substantial volume of RBCs and plasma are also collected in the procedure.
It is an object of the current invention to provide an improved collection procedure for harvesting sparse layers of stratified components in centrifuged liquids such as mononuclear cells in blood in order to collect a decreased volume with higher purity at high efficiency.